Sensitivity to toxicants is often enhanced during embryonic development, when exposure to low levels of chemicals can disrupt developmental process and lead to disease. Chemically-activated transcription factors act as environmental biosensors of externaltoxic stimuli and regulate gene expression involved in mitigating negative effects. Rarely is the response regulated by one transcription factor;rather, it is generally a coordinated effort between two or more transcription factors. One such interaction that may play an important role in the embryonic response to toxicant exposure is between two ligand-activated transcription factors -- the aryl hydrocarbon receptor (AHR) and nuclear factor erythroid 2-related factor 2 (NRF2). AHR and NRF2 independently regulate many genes involved in the cellular response to toxicants and oxidative stress as well as development, apoptosis, and differentiation. Because AHR and NRF2 regulate diverse gene targets, the significance of such crosstalk during development could be large;however, their crosstalk has only been characterized in adult mammals and cell culture. Zebrafish are an excellent model to study these interactions during development because of their rapid and external development as transparent embryos, the availability of extensive genomic and molecular tools, and conservation of developmental signaling pathways with humans, which can facilitate the extrapolation of results. This proposal is for research that uses a collaborative and multidisciplinary approach to elucidate AHR-NRF2 crosstalk and, in particular, the role of AHR in regulating transcription of NRF2 and its downstream genes during development and in response to toxicant exposure. Aim 1 seeks to understand the effects of AHR agonists on expression of NRF genes, and to determine which of the three zebrafish AHR genes regulate NRF expression. This will be investigated through the use of quantitative real-time PCR and morpholino antisense knockdown techniques. Aim 2 will determine the mechanism by which AHRs regulate the expression of NRF2 and other NRF genes. Through the use of bioinformatics, chromatin immunoprecipitation, and in vivo transient promoter assays, the cis- regulatory elements involved in AHR-dependent control of NRF expression will be identified. Aim 3 will determine the effect of AHR-NRF crosstalk on the expression and inducibility of NRF2 target genes. This will be accomplished by using qRT-PCR and microarray analysis, and through the measurement of reactive oxygen species. This research will provide a detailed and mechanistic understanding of the interaction of AHRs with NRFs. It will determine the importance of this interaction during the most sensitive life stage, the embryo, and provide better understanding of how combinatorial molecular signaling can protect embryos from potentially embryotoxic events following toxicant exposure. ) PUBLIC HEALTH RELEVANCE: Through the use of an in vivo animal model, the importance of two transcription factors involved in embryonic response to environmental toxicant exposure will be delineated. Because embryos are at increased risk from effects of toxicant exposure, it is important to understand the role of these transcription factors in both the pathogenesis of these effects as well as adaptive changes in gene expression that help to protect the embryo from toxicity.